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Abstract：
In vitro fertilization-embryo transfer (IVF-ET) is a crucial assisted reproductive technology for treating infertility. However, recurrent implantation failure (RIF), a significant challenge in IVF-ET success, remains unresolved. This study aimed to explore the role and mechanism of FLI1 in endometrial receptivity and RIF.
Differential endometrial cell proportions between patients with RIF and control subjects were assessed using single-cell RNA sequencing (scRNA-seq) analysis. The chromatin accessibility of FLI1 in the luteal endometrial tissue of patients with RIF and control subjects was examined using the single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). FLI1 mRNA and protein levels were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Cell viability and migration were examined via cell counting kit (CCK)-8 and scratch healing assays. Epithelial-mesenchymal transition markers were analyzed using western blotting. Mechanisms underlying FLI1\'s regulation of PART1 transcription and expression in endometrial epithelial cells were explored using chromatin immunoprecipitation and dual-luciferase reporter assays. Adeno-associated virus (AAV) carrying epithelial cell-specific FLI1/PART1 overexpression sequences was uterinely injected in mice to assess FLI1/PART1 effects.
scRNA-seq revealed diminished endometrial epithelial cell proportions in RIF patients. Meanwhile, scATAC-seq indicated enhanced chromatin accessibility of FLI1 in these cells. FLI1 exhibited specific expression in RIF patients\' endometrial epithelial cells. Specific FLI1 overexpression inhibited embryo implantation, while knockdown enhanced it. Pregnant mice injected with AAV encoding FLI1 overexpression had significantly lower implantation than AAV-negative controls. FLI1 binding to PART1 promoter heightened PART1 transcription and expression in endometrial epithelial cells. Rescue experiments illustrated FLI1\'s role in embryo implantation by boosting PART1 expression. PART1 was notably elevated in RIF patients\' luteal endometrial tissue and non-receptive endometrial epithelial cells (HEC-1-A). Specific PART1 overexpression dampened embryo implantation, whereas knockdown promoted it. Pregnant mice injected with AAV encoding PART1 had lower implantation than negative controls. PART1 knockdown mitigated FLI1\'s inhibitory impact on HEC-1-A cell viability and migration.
FLI1 overexpression in the endometrial epithelial cells of patients with RIF inhibited embryo implantation by binding to the PART1 promoter region to promote PART1 expression. These findings can aid in the development of novel therapeutic targets for RIF.
Differential endometrial cell proportions between patients with RIF and control subjects were assessed using single-cell RNA sequencing (scRNA-seq) analysis. The chromatin accessibility of FLI1 in the luteal endometrial tissue of patients with RIF and control subjects was examined using the single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). FLI1 mRNA and protein levels were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Cell viability and migration were examined via cell counting kit (CCK)-8 and scratch healing assays. Epithelial-mesenchymal transition markers were analyzed using western blotting. Mechanisms underlying FLI1\'s regulation of PART1 transcription and expression in endometrial epithelial cells were explored using chromatin immunoprecipitation and dual-luciferase reporter assays. Adeno-associated virus (AAV) carrying epithelial cell-specific FLI1/PART1 overexpression sequences was uterinely injected in mice to assess FLI1/PART1 effects.
scRNA-seq revealed diminished endometrial epithelial cell proportions in RIF patients. Meanwhile, scATAC-seq indicated enhanced chromatin accessibility of FLI1 in these cells. FLI1 exhibited specific expression in RIF patients\' endometrial epithelial cells. Specific FLI1 overexpression inhibited embryo implantation, while knockdown enhanced it. Pregnant mice injected with AAV encoding FLI1 overexpression had significantly lower implantation than AAV-negative controls. FLI1 binding to PART1 promoter heightened PART1 transcription and expression in endometrial epithelial cells. Rescue experiments illustrated FLI1\'s role in embryo implantation by boosting PART1 expression. PART1 was notably elevated in RIF patients\' luteal endometrial tissue and non-receptive endometrial epithelial cells (HEC-1-A). Specific PART1 overexpression dampened embryo implantation, whereas knockdown promoted it. Pregnant mice injected with AAV encoding PART1 had lower implantation than negative controls. PART1 knockdown mitigated FLI1\'s inhibitory impact on HEC-1-A cell viability and migration.
FLI1 overexpression in the endometrial epithelial cells of patients with RIF inhibited embryo implantation by binding to the PART1 promoter region to promote PART1 expression. These findings can aid in the development of novel therapeutic targets for RIF.
摘要：
■体外受精-胚胎移植（IVF-ET）是治疗不孕症的重要辅助生殖技术。然而,复发性植入失败（RIF），IVF-ET成功的重大挑战，仍未解决。本研究旨在探讨FLI1在子宫内膜容受性和RIF中的作用及其机制。
使用单细胞RNA测序(scRNA-seq)分析评估RIF患者和对照受试者之间的子宫内膜细胞比例差异。使用转座酶可接近染色质测序的单细胞测定法（scATAC-seq）检查了RIF患者和对照受试者黄体子宫内膜组织中FLI1的染色质可接近性。通过定量实时聚合酶链反应(qRT-PCR)和蛋白质印迹测定FLI1mRNA和蛋白质水平。通过细胞计数试剂盒(CCK)-8和划痕愈合测定检查细胞活力和迁移。使用蛋白质印迹分析上皮-间质转化标志物。利用染色质免疫沉淀法和双荧光素酶报告基因分析，探讨了FLI1调节子宫内膜上皮细胞PART1转录和表达的潜在机制。将携带上皮细胞特异性FLI1/PART1过表达序列的腺相关病毒(AAV)子宫内注射到小鼠中以评估FLI1/PART1效应。
■scRNA-seq显示RIF患者子宫内膜上皮细胞比例减少。同时,scATAC-seq表明这些细胞中FLI1的染色质可及性增强。FLI1在RIF患者子宫内膜上皮细胞中表现出特异性表达。特异性FLI1过表达抑制胚胎着床，而击倒增强了它。注射编码FLI1过表达的AAV的妊娠小鼠的植入明显低于AAV阴性对照。FLI1与PART1启动子的结合增强了子宫内膜上皮细胞中PART1的转录和表达。挽救实验通过增强PART1表达说明了FLI1在胚胎植入中的作用。PART1在RIF患者黄体子宫内膜组织和非接受性子宫内膜上皮细胞（HEC-1-A）中显著升高。特异性PART1过表达抑制胚胎植入，而击倒促进了它。注射编码PART1的AAV的妊娠小鼠的植入低于阴性对照。PART1敲低减轻FLI1对HEC-1-A细胞活力和迁移的抑制作用。
■FLI1在RIF患者子宫内膜上皮细胞中的过表达通过结合PART1启动子区促进PART1表达来抑制胚胎着床。这些发现可以帮助开发RIF的新治疗靶标。
使用单细胞RNA测序(scRNA-seq)分析评估RIF患者和对照受试者之间的子宫内膜细胞比例差异。使用转座酶可接近染色质测序的单细胞测定法（scATAC-seq）检查了RIF患者和对照受试者黄体子宫内膜组织中FLI1的染色质可接近性。通过定量实时聚合酶链反应(qRT-PCR)和蛋白质印迹测定FLI1mRNA和蛋白质水平。通过细胞计数试剂盒(CCK)-8和划痕愈合测定检查细胞活力和迁移。使用蛋白质印迹分析上皮-间质转化标志物。利用染色质免疫沉淀法和双荧光素酶报告基因分析，探讨了FLI1调节子宫内膜上皮细胞PART1转录和表达的潜在机制。将携带上皮细胞特异性FLI1/PART1过表达序列的腺相关病毒(AAV)子宫内注射到小鼠中以评估FLI1/PART1效应。
■scRNA-seq显示RIF患者子宫内膜上皮细胞比例减少。同时,scATAC-seq表明这些细胞中FLI1的染色质可及性增强。FLI1在RIF患者子宫内膜上皮细胞中表现出特异性表达。特异性FLI1过表达抑制胚胎着床，而击倒增强了它。注射编码FLI1过表达的AAV的妊娠小鼠的植入明显低于AAV阴性对照。FLI1与PART1启动子的结合增强了子宫内膜上皮细胞中PART1的转录和表达。挽救实验通过增强PART1表达说明了FLI1在胚胎植入中的作用。PART1在RIF患者黄体子宫内膜组织和非接受性子宫内膜上皮细胞（HEC-1-A）中显著升高。特异性PART1过表达抑制胚胎植入，而击倒促进了它。注射编码PART1的AAV的妊娠小鼠的植入低于阴性对照。PART1敲低减轻FLI1对HEC-1-A细胞活力和迁移的抑制作用。
■FLI1在RIF患者子宫内膜上皮细胞中的过表达通过结合PART1启动子区促进PART1表达来抑制胚胎着床。这些发现可以帮助开发RIF的新治疗靶标。
